Oomycetes associated withsoybean disease and improved

diagnosticsMartin Chilvers & OSCAP Extension

Networkchilvers@msu.edu

@MartinChilvers1517-353-9967

www.fieldcroppathology.msu.edu

Integrated management of oomycetediseases of soybean and other crop

plants§ Survey oomycetes associated with diseasedsoybean seedlings

§ Characterize pathogenicity and aggressiveness§ Determine fungicide sensitivity – high throughput§ Develop diagnostics for improved management

Two year survey conducted in conjunction with OSCAP network• Which oomycetes are associated with soybean seedling disease?• Are oomycete communities similar across locations?

• Isolation from diseased seedlings

Soybean seedling disease survey

• 84 unique species found ~3200 cultures• Pathogenicity/Virulence assessed for a subset of isolates across

all species– Seed rot assay– Seedling root rot assay

Survey of oomycete species Results

1 Aphanomyces spp.

2 Phytopythium spp.

3 Phytophthora spp.

52 Pythium spp.

4 Phytopythium spp.

7 Phytophthora spp.

57 Pythium spp.

Oomycetes community structure

• States were clustered based on relative abundance of species• Latitude is correlated with species richness• Temperature and precipitation were main drivers of community

structure

2011

• Many beneficial species• Which species cause

disease?• Does plant growth stage

affect susceptibility? • What effect does

temperature have ondisease?

• Two different methods:– Seed rot– Seedling root rot

Seed Rot Assays

Pathogen +Seed

Severity0 Germinated1 Delayed germination2 Germination and some lesions3 Germination with coalesced lesions4 Seed colonized

DSI = Σ(severity X n) N

Disease Index =(DSI)

7days in dark

13C and 20C

Broders et al., 2007

Pythium oopapillum Pythium irregulareControl Pythium ultimum var.ultimum

Phytophthora sojae Pythium sylvaticum Pythium attrantheridium Pythium ultimum var.sporangiiferum

Seed Rot Assays

Seed rot severity index DSI at 20oC (68oF)

P. sojae

Py. sylvaticum

Seed rot DSI – 13oC (55oF) vs 20oC (68oF)

• P. oopapillum prevalent in cooler 2011 – more pathogenic at 55oFthan 68oF

• P. sojae not very aggressive on seed, slightly more at warmer temp.

13C

20C

Seedling root rot assays

• Dry weight of roots and shoots• Root area and root length

Pythium oopapillumControl Pythium irregulare Pythium ultimum var.ultimum

Phytophthora sojae Pythium sylvaticum Pythium attrantheridium Pythium ultimum var.sporangiiferum

Seedling root rot assays

Variability in seedling root rot at20oC

Me

an r

oot w

eigh

t

P. sojaeP. sylvaticum

Molecular diagnostics• Different end users

– Different requirements

• Hierarchicalapproach

– Genus • Phytophthora

– Species• P. sojae• P. sansomeana

• Testing andvalidation

– Specificity andsensitivity

– Culture collection

Quantitative PCRHydrolysis probe

Isothermal PCR

Amplicon-basedcommunity analysis

Chilvers 2012. Fung. Genom. & Biol. 2:e102.

Phytophthora sojae– Well recognized– Narrow host range – soybean, lupin

Phytophthora sansomeana (described 2009)– Not as well recognized– Wide host range - soybean, corn, Douglas-fir

Photo Credit: Anette Phibbs, DATCP.

Phytophthora sojae Phytophthora sansomeana

Multiplex qPCR for P. sojae and P. sansomeana

collaboration with Frank Martin and Tim Miles

• Multiplex assay for detection of two species– Plant samples (Plant internal control)– Soil samples (Internal control)

Phytophthora genus specif ic probe

P. sojaespecies specif ic probe

P. sansomeanaspecies specif ic probePrimer F Primer R

Bilodeau et al. Phytopath. 104:733

Validation of qPCR assaySpecificity test panel• 96 different Phytophthora spp.• 14 provisional Phytophthora species• 10 Pythium spp.

Genus

P. sojae

Isothermal amplificationRecombinase Polymerase Amplification

(RPA)

Smart Dart (BioRanger)

The RPA Cycle

RPA advantages• All steps at

37ºC • Recombinase

opens dsDNA• End point or

real-time

Detection in ~ 20 minutes!

http://www.twistdx.co.uk/our_technology/

RPA process: P. sojae and P. sansomeana

Isothermal RPA - Sample Results

Real-time data collection End-point diagnostic data

Positive Negative

Control Band

Positive Rxn

Amplicon basedcommunity analysis• Oomycetes poorly represented in

metagenomes• But play important role in microbial

ecology• Pair w/ phenotype data

Amplicon vs Culture

Oomycetes - Summary and futurework

• Oomycete species vary by region• 84 unique oomycete species

associated with soybeanseedlings– 17 pathogenic in seed rot assay– 43 pathogenic in seedling assay– 15 species pathogenic in both

assays• Hierarchical qPCR and RPA

diagnostic assays • Which chemistries are most

effective? • Breeding for resistance – Dechun

Wang and Yingdong Bi

Acknowledgements• OSCAP Extension Network:Carl Bradley, Tom Chase, Paul Esker, Loren Giesler, Doug Jardine, Dean Malvick, Sam Markell, Berlin Nelson, Alison Robertson, John Rupe, Damon Smith, Laura Sweets, Albert Tenuta, Kiersten Wise• MSU diagnostic lab• John Boyse and Randy

LaurenzProject No. 2011-68004-30104 (“Integrated management of oomycete diseases of soybean and other crop plants”)

Oomycete SurveySummary • 84 unique oomycete species associated with soybean

seedlings

• Oomycete community structure correlated with geographicalproximity

• Oomycete species abundance varied by latitude

• 17 species were pathogenic in the seed rot assay

• 43 species pathogenic on seedlings for at least oneparameter

• 15 species were pathogenic in both seed and seedlingassays

Diagnostic Assay Summary • Multiplex hierarchical qPCR assay for

Phytophthora genus, P. sojae, P. sansomeana

• Implemented by diagnosticians

• Hierarchical RPA assay for Phytophthora,P. sojae, P. sansomeana

• qPCR and RPA validated with fieldsamples from 8 U.S. states and Ontario

Oomycete community structure

• Samples analyzed based on relative abundance of the species• Temperature and precipitation were main drivers of community structure

Seedling pathogenicity

Fungi associated with diseased soybean seedlings

Courtesy Ahmad Fakoury, Univ. of Southern Illinois

3000 isolates

Fusarium species

Courtesy Ahmad Fakoury, Univ. of Southern Illinois

• Roughly 70% of soybeans aretreated (Munkvold 2009)

• Critical to understand fungicidesensitivity for best management

• Sensitivity of species recoveredin survey not well understood

Fungicide sensitivity Introduction

Fungicides used in seed treatmentsWhat is mefenoxam and ethaboxam?

Metalaxyl

Mefenoxam

Ethaboxam

RNA Polymerase 1

FRAC group A1 FRAC group B3

β-tubulin assembly – Uchida et al. 2005

Well documented resistance in oomycetes Registered for soybean seed treatments 2014

High-throughput fungicide sensitivity Assay development

¼ strength PCA 0.5% agar

5 or 6, 16.5 mm plugs

2 - 5 day old cultures

1. Macerate Agar

Vortex for 10 seconds

0 � g ml-1

Optical density 600nm

Tecan Plate

reader

24 - 48 hours growth

½ strength V8 broth + Fungicide

50�L

2. Distribute to 96 Well

10 ml syringe

20G needle

mefenoxam

ethaboxam

0.01 � g ml-1

0.1 � g ml-1

0.5 � g ml-1

1 � g ml-1

10 � g ml-1

0 � g ml-1

0.01 � g ml-1

0.1 � g ml-1

0.5 � g ml-1

1 � g ml-1

5 � g ml-1

20 � g ml-1

0 � g ml-1

Growth Curves

3. Measure

ControlsInoculated controlNon-inoculated control½ strength V8 broth

High-throughput fungicide sensitivity Assay validation

35

Species0. Phytophthora sansomeana1. Pythium aff. dissotocum2. Pythium aphanidermatum3. Pythium irregulare4. Pythium lutarium5. Pythium oopapillum6. Pythium perplexum7. Pythium sylvaticum8. Pythium torulosum9. Pythium ultimum var.

ultimumR2 = 0.88Pearson’s r = 0.94

Ethaboxam – P � 0.05

High-throughput fungicide sensitivity Community inference

20 � g ml-1 Ethaboxam

10 � g a.i. ml-1 mefenoxam

20 � g ml-1 ethaboxam

• High throughput ~4 hours set up 30 isolates,multiple chemistries

• Poison plate ~1 day set up 12 isolates, onechemistry

• Most species appear sensitive to mefenoxam

• Ethaboxam insensitivity conserved amongstmonophyletic groups

Fungicide Sensitivity Summary

Oomycete resources• Databases!

– Curated and consolidated database– Joint effort among the community

• Taxonomy– Species?– Consolidated taxonomy

• A. Levesque and AW de Cock • F. Martin and J. Blair (2014)

� Hyde et al. 2014

Phytophthora spp.Pythium spp.